A number of antibiotic compounds have been developed and shown to be effective in inhibiting translation. The antibiotics neomycin, thiostrepton, and hygromycin appear to inhibit translocation and occupation of the A site within a ribosome but have no effect on formation of the fMet-tRNAfMet/ribosome translation complex nor on the peptide-bond synthesis which occurs in the absence of elongation factor P. Streptomycin, which causes misreading and also inhibits A-site binding, interacts with two sites on the 16S rRNA of the 30S subunit. Lincomycin inhibits peptidyltransferase and occupation of the A-site. Erythromycin inhibits peptidyltransferase and destabilizes the peptidyl-tRNA/ribosome/mRNA complex.
A number of additional compounds have been recently developed and have been shown to act as antimicrobial or antibacterial agents. International Publication WO 99/41244 discloses substituted aminophenyl isoxazoline compounds useful as antimicrobial agents. U.S. Pat. No. 5,910,504 describes hetero-aromatic ring substituted phenyloxazolidinone antimicrobial agents. In addition, International Publication WO 00/10566 discloses isoxazolinone antibacterial agents. An important step in the development of new antimicrobial or antibacterial agents, such as those disclosed above, is the elucidation of a mechanism of action. The specific site of interaction of non selective antibiotics/antitumor agents, such as sparsomycin, that inhibit protein translation by a different, less useful, and direct mechanism, has been described. Porse et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 9003-9008. Previous studies with chemical probes using isolated, cell-free systems have failed to define the relative sites of interaction of these types of antibiotic compounds of the oxazolidinone class. Matassova, et al., RNA, 1999, 5, 939-946. This is, in part, because the previous methods were incapable of defining the sites of the particular and specific mechanism of action of this important class of antibiotics. Probes that help to elucidate the mechanism of action of antimicrobial and/or antibacterial agents and methods of using the same are highly desired.
The present invention is directed, inter alia, to novel oxazolidinone compounds and use of oxazolidinone compounds as photoaffinity probes. Compounds suspected of having antimicrobial and/or antibacterial activity can be used in intact cells and can be evaluated for a mechanism of action by using active and inactive enantiomers of oxazolidinone compounds as competitors for crosslinking to components within the cell. The compounds and methods of the present invention allow one skilled in the art to elucidate the mechanism of action of antibacterial and/or antimicrobial agents that are suspected of inhibiting protein translation. These and other aspects of the invention are described below.